A LAT1-Like Amino Acid Transporter Regulates Neuronal Activity in the Drosophila Mushroom Bodies

The proper functioning of neural circuits that integrate sensory signals is essential for individual adaptation to an ever-changing environment. Many molecules can modulate neuronal activity, including neurotransmitters, receptors, and even amino acids. Here, we ask whether amino acid transporters expressed by neurons can influence neuronal activity. We found that minidiscs (mnd), which encodes a light chain of a heterodimeric amino acid transporter, is expressed in different cell types of the adult Drosophila brain: in mushroom body neurons (MBs) and in glial cells. Using live calcium imaging, we found that MND expressed in α/β MB neurons is essential for sensitivity to the L-amino acids: Leu, Ile, Asp, Glu, Lys, Thr, and Arg. We found that the Target Of Rapamycin (TOR) pathway but not the Glutamate Dehydrogenase (GDH) pathway is involved in the Leucine-dependent response of α/β MB neurons. This study strongly supports the key role of MND in regulating MB activity in response to amino acids.


Introduction
For any living organism, such as Drosophila melanogaster, an adequate supply of nutrients, especially amino acids (AAs), is essential to ensure a variety of functions including development [1], growth [2], lifespan and survival [3][4][5], reproduction [6,7], egg production and fecundity [3,4,8], and sleep [9].While some AAs can be synthesized endogenously from precursors, others, known as essential amino acids (EAAs), such as Leu, Ile, Thr, are supplied by the diet.They also provide energy to fuel cellular metabolism.Some AAs are precursors of hormones, such as tryptophan and tyrosine, which are used to produce melatonin and noradrenaline/adrenaline, respectively [10,11].Some AAs are also neurotransmitter precursors, such as tryptophan, which is required for serotonin production; tyrosine is required for L-dopa and dopamine production [12][13][14].Others, such as glutamate, are used directly as neurotransmitters [15], and glutamate is also the precursor of the neurotransmitter GABA [16].
Mammals and invertebrates, such as Drosophila, are able to maintain an appropriate AA balance by selecting diets that contain the EAAs they need and avoiding diets that lack EAAs [17,18].In Drosophila larvae, an unbalanced AA diet is detected by dopaminergic neurons (DANs) in the brain via an intracellular AA sensor: the serine/threonine kinase General Control Nonderepressible 2 (GCN2) which acts upstream of GABA signaling to inhibit it and promotes avoidance of an EAA-deficient diet [18].In adults, food intake is also promoted by three AAs: Glu, Asp, and Ala [19].EAAs such as Leu and Ile are required for the release of insulin-like peptides (DILPs) by insulin-producing cells (IPCs) in the brain via a GDH pathway to ensure proper larval metabolism [20,21].Methionine-supplemented diets reduce survival but have no effect on reproduction [22], whereas reduced methionine intake extends lifespan and reduces reproduction in low AA status [23,24].Threonine, another EAA, is a sleep-promoting molecule that links neuronal metabolism to GABAergic control of sleep.Thus, threonine may be the neuronal substrate for sleep homeostasis [9].
In the adult Drosophila brain, the mushroom bodies (MBs), an integrative brain center, are a critical structure for appetitive olfactory learning and memory [25][26][27][28], and aversive olfactory memory [29,30].In addition, the integration of metabolic cues by the MBs has a critical impact on the control of learned behaviors [31][32][33].The MBs consist of 2000 to 2500 neurons per hemisphere [34], called Kenyon cells [35], which project their axons into two (α and α ′ ) vertical lobes and three (β, β ′ , and Cells 2024, 13, x FOR PEER REVIEW 2 o (IPCs) in the brain via a GDH pathway to ensure proper larval metabolism [20,21].Met onine-supplemented diets reduce survival but have no effect on reproduction [2 whereas reduced methionine intake extends lifespan and reduces reproduction in low A status [23,24].Threonine, another EAA, is a sleep-promoting molecule that links neuro metabolism to GABAergic control of sleep.Thus, threonine may be the neuronal substr for sleep homeostasis [9].
In the adult Drosophila brain, the mushroom bodies (MBs), an integrative brain cen are a critical structure for appetitive olfactory learning and memory [25][26][27][28], and avers olfactory memory [29,30].In addition, the integration of metabolic cues by the MBs ha critical impact on the control of learned behaviors [31][32][33].The MBs consist of 2000 to 25 neurons per hemisphere [34], called Kenyon cells [35], which project their axons into t (α and α′) vertical lobes and three (β, β′, and ɣ) medial lobes [34,36].Kenyon cells rece olfactory inputs from the antennal lobes via projection neurons, process the informati and enable olfactory associative learning [37] and memory [38].Numerous other stim such as visual, thermosensory, and gustatory inputs [39][40][41][42][43][44], are delivered to the M placing this structure at the center of signal integration and behaviors.In addition, the M α/β lobes are associated with lifespan [45], which is directly influenced by the quality a quantity of the diet and EAA intake [45].
In this study, we investigate the function of MND in the response to AAs in the ad brain.First, we show that mnd is expressed in both glial cells and neurons in the ad Drosophila brain.By generating specific Gal4 and LexA reporter transgenes, we identifi two distinct regulatory regions in the mnd gene that control expression either in the mu room body neurons (α/β and ɣ lobe neurons of the MBs) or in cortex glia.We used MB-specific mnd driver to study the neuronal activity in response to different AAs in vivo functional brain imaging and revealed that MB neurons respond to a wide range AAs.Here, we show that mnd knockdown in α/β MB lobes results in impaired respon to several AAs, including Leu, Ile, Arg, Asp, Glu, Lys, and Thr.Finally, we found t Leucine, an EAA, activates the MBs through the TOR pathway rather than a GDH sign ing pathway.Our data establish that MBs are an important brain center for internal A sensing that depends on the presence of MND, and this highlights how amino acid tra porters, such as SLC family transporters, can influence neuronal activity, which may p vide new clues to better understand the regulation of neuronal activity.

Drosophila Strains
All Drosophila melanogaster strains were maintained on standard cornmeal/yeast/a medium, at 25 °C, on a 12 h:12 h light-dark cycle, with 50-60% relative humidity.The strains used in this study were as follows: w 1118 , mnd 24−1 -Gal4, mnd 25−1 -Gal4, and mnd 2   ) medial lobes [34,36].Kenyon cells receive olfactory inputs from the antennal lobes via projection neurons, process the information, and enable olfactory associative learning [37] and memory [38].Numerous other stimuli, such as visual, thermosensory, and gustatory inputs [39][40][41][42][43][44], are delivered to the MBs, placing this structure at the center of signal integration and behaviors.In addition, the MB α/β lobes are associated with lifespan [45], which is directly influenced by the quality and quantity of the diet and EAA intake [45].
AA transporters, particularly members of the Solute Carrier family (SLC), play a fundamental role in AA transport in cells, including neurons [46][47][48].SLC transporters are divided into several families, particularly the SLC7A family which is divided into two subfamilies: the Cationic Amino-acid Transporters (CATs) [49], and the L-type Amino-acid Transporters (LATs) [50].In Drosophila, a specific member of the CAT family, named Slimfast, is expressed in larval fat body cells and in adult DANs, allowing the activation of TOR [51,52] and the AA sensor GCN2 [18], respectively.This activation leads to the regulation of growth and food intake [18,51].While CATs, such as Slimfast, function as monomers, LATs form Heterodimeric Amino-acid Transporters (HATs) [50,53].These HATs consist of two subunits, a heavy chain (SLC3A2/CD98hc) and a light chain [54], encoded by five putative genes in Drosophila (minidiscs, JhI-21, genderblind, sobremesa and CG1607) [55][56][57][58].In mammals, SLC3A2/CD98/4F2hc targets the complex to the plasma membrane, and the light chain determines the specificity of the AA transporter [59,60].In Drosophila S2 cells, a light chain called Minidiscs (MND) is required for leucine transport [61].
In this study, we investigate the function of MND in the response to AAs in the adult brain.First, we show that mnd is expressed in both glial cells and neurons in the adult Drosophila brain.By generating specific Gal4 and LexA reporter transgenes, we identified two distinct regulatory regions in the mnd gene that control expression either in the mushroom body neurons (α/β and (IPCs) in the brain via a GDH pathway to ensure proper larval metabolism [20,21].Methionine-supplemented diets reduce survival but have no effect on reproduction [22], whereas reduced methionine intake extends lifespan and reduces reproduction in low AA status [23,24].Threonine, another EAA, is a sleep-promoting molecule that links neuronal metabolism to GABAergic control of sleep.Thus, threonine may be the neuronal substrate for sleep homeostasis [9].
In the adult Drosophila brain, the mushroom bodies (MBs), an integrative brain center, are a critical structure for appetitive olfactory learning and memory [25][26][27][28], and aversive olfactory memory [29,30].In addition, the integration of metabolic cues by the MBs has a critical impact on the control of learned behaviors [31][32][33].The MBs consist of 2000 to 2500 neurons per hemisphere [34], called Kenyon cells [35], which project their axons into two (α and α′) vertical lobes and three (β, β′, and ɣ) medial lobes [34,36].Kenyon cells receive olfactory inputs from the antennal lobes via projection neurons, process the information, and enable olfactory associative learning [37] and memory [38].Numerous other stimuli, such as visual, thermosensory, and gustatory inputs [39][40][41][42][43][44], are delivered to the MBs, placing this structure at the center of signal integration and behaviors.In addition, the MB α/β lobes are associated with lifespan [45], which is directly influenced by the quality and quantity of the diet and EAA intake [45].
In this study, we investigate the function of MND in the response to AAs in the adult brain.First, we show that mnd is expressed in both glial cells and neurons in the adult Drosophila brain.By generating specific Gal4 and LexA reporter transgenes, we identified two distinct regulatory regions in the mnd gene that control expression either in the mush-lobe neurons of the MBs) or in cortex glia.We used the MB-specific mnd driver to study the neuronal activity in response to different AAs in ex vivo functional brain imaging and revealed that MB neurons respond to a wide range of AAs.Here, we show that mnd knockdown in α/β MB lobes results in impaired responses to several AAs, including Leu, Ile, Arg, Asp, Glu, Lys, and Thr.Finally, we found that Leucine, an EAA, activates the MBs through the TOR pathway rather than a GDH signaling pathway.Our data establish that MBs are an important brain center for internal AA sensing that depends on the presence of MND, and this highlights how amino acid transporters, such as SLC family transporters, can influence neuronal activity, which may provide new clues to better understand the regulation of neuronal activity.

gDNA Extraction
Adult flies were frozen and ground in a buffer (0.1 M EDTA pH 8; 0.1 M Tris-HCl pH 9; SDS 1%), incubated for 30 min at 70 • C, and then kept on ice for 30 min after the addition of 28 µL potassium acetate solution 8 M.After centrifugation at 14,000× g for 10 min at 4 • C, isopropanol was added to precipitate nucleic acids.After 10 min at −80 • C and centrifugation at 14,000× g for 5 min at 4 • C, gDNA was washed twice with 70% ethanol and dissolved in Tris EDTA pH8.

Whole-Mount Immunostaining
Adult brains were dissected in PBS, fixed in 4% paraformaldehyde for 45 min at room temperature (RT), and washed for 6 × 10 min in PBS + 0.3% Triton X-100 (PBS-T) and 1 × 10 min in PBS + 1%Triton X-100 to permeabilize membranes.Tissues were blocked for 1 h at RT in PBS-T containing 10% normal goat serum (NGS; Sigma #G9023).Appropriate primary antibodies were diluted in PBS-T + 5% NGS and incubated with the tissues for 1 day at 4 • C.After washing 6 × 10 min in PBS-T, samples were labeled with the appropriate secondary antibody at 1:400 in PBS-T containing 5% NGS for 3 h at RT.They were washed for 6 × 10 min in PBS and mounted in VECTASHIELD ® mounting medium (H-1000, Vector Laboratories, Peterborough, UK).Fluorescence was observed with a Leica TCS SP8 confocal microscope.

Cross-Sectioning of Adult Brains
For sectioning, the proboscis, wings, and legs were removed from adult Drosophila bodies and fixed in 4% paraformaldehyde (PFA) in PBS pH 7.2 for 3 h at 4 • C. The fixative was then replaced with 25% sucrose in Drosophila Ringer's solution (46 mM NaCl, 182 mM KCl, 3 mM CaCl 2 , 10 mM Tris-HCl, pH 7.2) and incubated overnight at 4 • C. The heads were excised, embedded in Tissue-Tek (Sakura Finetek, Torrance, CA, USA), frozen in liquid nitrogen, and sectioned at 14 µm using a cryostat (Leica CM 3050).Sections were washed 2 × 10 min with TBS + 2.5% Triton (TBS-T) and blocked with 5% NGS for 30 min at RT.Primary antibodies were diluted in a blocking solution and incubated with the samples for 1 day at 4 • C. Sections were washed 2 × 10 min with TBS-T and incubated with the appropriate secondary antibodies diluted in a blocking solution and incubated with the samples overnight at 4 • C. Sections were washed 2 × 10 min at RT and mounted in VECTASHIELD ® Hardest TM mounting medium.Fluorescence was observed with a Leica TCS SP8 confocal microscope.

Fluorescence Quantification
Immunohistochemical analysis of the MND protein level within the Kenyon cells of control brains (mnd 24−1 -Gal4 > UAS-mCD8::GFP) and mnd downregulated brains (mnd 24−1 -Gal4 > UAS-mCD8::GFP) was performed.Confocal images were obtained using a 63× objective using a 1 µm step size, and the same laser power and scanning settings were used for all samples, using a Leica TCS SP8 confocal microscope.Mean MND fluorescence intensity in the Kenyon cells was quantified from confocal z-stack images using FIJI software (ImageJ 1.47 k).The signal from an adjacent region to the Kenyon cells served as an autofluorescence background and was subtracted from the mean MND fluorescence in these neurons.To compare the two genotypes, MND values of control brains (mnd 24−1 -Gal4 > UAS-mCD8::GFP) were used as a reference (immunofluorescence = 1).

Calcium Imaging
For ex vivo live calcium imaging experiments, 3-day-old adults were immobilized on ice and the brains were carefully and rapidly removed from the capsule head in Ringer's saline (130 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 36 mM saccharose, 5 mM HEPES, pH 7.3) and placed on a silicone plate in a 150 µL drop of Ringer's saline.To prevent any movement during the imaging, the brains were fixed to the silicone support with insect pins in the optic lobes.GCaMP6s fluorescence was recorded using a Leica DM6000B microscope under a 25× water objective.GCaMP6s was excited using a Lumencor light engine supplied with diodes at 485 ± 25 nm.The emitted light was collected through a 505-530 nm bandpass filter.Leica MM AF 2.2.0 was used for data collection and acquisition.Images were acquired at 500 ms per frame at 256 × 256 resolution using an Orca-Flash 4.0 camera.A minimum of 480 images were acquired for each experiment: 100 before and 380 after the AA application.The first ten frames before the AA application were used to establish the baseline F 0 .Regions adjacent to the region of interest were used to determine the autofluorescence background level.Changes in fluorescence from initial fluorescence (∆F/F 0 ) were calculated as the peak fluorescence after t = 100 frames minus F 0 versus F 0 , [64].AA solutions are extemporaneously prepared with an appropriate concentration in Ringer's saline.All L-AAs used in this study were purchased from Sigma-Aldrich.

Statistical Analysis
Prism 9 software (Graphpad, v9.0, Boston, MA, USA) was used for statistical analyses.Shapiro-Wilk and D'Agostino-Pearson tests were used to assess normality for all individual experiments.The two-tailed unpaired Student's t-test was used for comparison between two groups with normally distributed data, and the Mann-Whitney test was used for data that did not pass the normality test.For comparisons between more than two groups with normally distributed data, one-way ANOVA was used, and the Kruskall-Wallis test was used for data that did not pass the normality test.

mnd Is Expressed in the Adult Drosophila Brain
We first examined the expression pattern of mnd in adult Drosophila.Three mnd transcripts were predicted.All should derive through alternative splicing of the 5 ′ UTR, generate mnd-RA, mnd-RC, and mnd-RD, which all encode the same protein [55], see Figure 1a.According to the Flybase prediction (FB2024_03, released 25 June 2024), mnd may be expressed in different tissues including the adult brain.To test whether these splice forms may be present in different tissues, we first attempted to detect these three mnd transcripts by RT-PCR in adult heads and bodies.We designed specific primers for each cDNA of mnd transcripts, represented by colored arrows in Figure 1a.After separating the RT-PCR fragments by agarose gel electrophoresis, we found that mnd-RA (111 pb) and mnd-RC (183 pb) are expressed in the heads and the bodies, while mnd-RD (114 pb) is not (Figure 1b), indicating that mnd-RD may be very weakly expressed or does not exist as predicted.The amplified fragment for each mRNA has been sequenced and confirmed the expression of the three different transcripts.
Next, we also investigated MND expression using MND antibodies [20], and we showed that MND is broadly expressed in the brain (Figure S1a) and that the antibodies are specific to MND (Figure S1c,d).To identify the type of cells expressing MND, we used GFP-expression driven by a neuronal-specific driver (elav-Gal4) or a glia-specific driver (repo-Gal4), the two major cell types in the brain.MND was detected in both neurons and glial cells (Figure 1c).

The Regulatory Promoter Sequences of Mnd Specifically Drives Expression either in Neurons or in Glial Cells
We then attempted to identify mnd regulatory sequences capable of driving expression in the nervous system by generating reporter transgenic lines.By bioinformatic analysis, we found two potential regulatory sequences illustrated by the blue box for the region called mnd 24−1 and the green box for the mnd 25−1 region in Figure 2a.
We generated a Gal4 driver transgene, mnd 25−1 -Gal4, containing the regulatory sequence of mnd corresponding to the 5′UTR region of the second mnd exon (Figure 2a).This transgene drives expression in the adult brain, particularly in glial cells (Figure 2b).
We then found that the mnd 24−1 -Gal4 transgene containing the regulatory sequences of the 5′ region of the first mnd exon (Figure 2a) drives expression in the brain, particularly in the neurons of the MBs (Figure 2(c1)).Specifically, we observed that mnd 24−1 -Gal4 driver expression in the MBs appeared to be restricted to certain lobes.We performed immunostaining on mnd 24−1 -Gal4 > UAS-mCD8::GFP brains to examine the presence of MND

The Regulatory Promoter Sequences of Mnd Specifically Drives Expression either in Neurons or in Glial Cells
We then attempted to identify mnd regulatory sequences capable of driving expression in the nervous system by generating reporter transgenic lines.By bioinformatic analysis, we found two potential regulatory sequences illustrated by the blue box for the region called mnd 24−1 and the green box for the mnd 25−1 region in Figure 2a.
We then found that the mnd 24−1 -Gal4 transgene containing the regulatory sequences of the 5 ′ region of the first mnd exon (Figure 2a) drives expression in the brain, particularly in the neurons of the MBs (Figure 2(c1)).Specifically, we observed that mnd 24−1 -Gal4 driver expression in the MBs appeared to be restricted to certain lobes.We performed immunostaining on mnd 24−1 -Gal4 > UAS-mCD8::GFP brains to examine the presence of MND (Figure 2(c1 ′′ ,c1**)).We observed that MND and GFP co-localized in the cell body of the Kenyon cells (Kcs) (Figure 2(c1*,c1**,c1***)) and found weak MND expression in the MB calyx but not in MB lobes (Figure 2(c1 ′ ,c1 ′′ ,c1 ′′′ )).This supports that the neuronal expression of MND is under the control of the specific mnd 24−1 regulatory sequence.
The MBs are constituted by about 2000 and 2500 Kenyon cells per hemisphere, which send their axons in different directions forming three different lobes, the α/β, α ′ /β ′ , and 2 of 18 n the brain via a GDH pathway to ensure proper larval metabolism [20,21].Methiupplemented diets reduce survival but have no effect on reproduction [22], s reduced methionine intake extends lifespan and reduces reproduction in low AA 3,24].Threonine, another EAA, is a sleep-promoting molecule that links neuronal lism to GABAergic control of sleep.Thus, threonine may be the neuronal substrate homeostasis [9]. the adult Drosophila brain, the mushroom bodies (MBs), an integrative brain center, tical structure for appetitive olfactory learning and memory [25][26][27][28], and aversive y memory [29,30].In addition, the integration of metabolic cues by the MBs has a mpact on the control of learned behaviors [31][32][33].The MBs consist of 2000 to 2500 per hemisphere [34], called Kenyon cells [35], which project their axons into two ′) vertical lobes and three (β, β′, and ɣ) medial lobes [34,36].Kenyon cells receive y inputs from the antennal lobes via projection neurons, process the information, ble olfactory associative learning [37] and memory [38].Numerous other stimuli, visual, thermosensory, and gustatory inputs [39][40][41][42][43][44], are delivered to the MBs, this structure at the center of signal integration and behaviors.In addition, the MB s are associated with lifespan [45], which is directly influenced by the quality and of the diet and EAA intake [45].transporters, particularly members of the Solute Carrier family (SLC), play a funal role in AA transport in cells, including neurons [46][47][48].SLC transporters are into several families, particularly the SLC7A family which is divided into two ilies: the Cationic Amino-acid Transporters (CATs) [49], and the L-type Aminonsporters (LATs) [50].In Drosophila, a specific member of the CAT family, named t, is expressed in larval fat body cells and in adult DANs, allowing the activation [51,52] and the AA sensor GCN2 [18], respectively.This activation leads to the on of growth and food intake [18,51].While CATs, such as Slimfast, function as ers, LATs form Heterodimeric Amino-acid Transporters (HATs) [50,53].These nsist of two subunits, a heavy chain (SLC3A2/CD98hc) and a light chain [54], eny five putative genes in Drosophila (minidiscs, JhI-21, genderblind, sobremesa and ) [55][56][57][58].In mammals, SLC3A2/CD98/4F2hc targets the complex to the plasma ne, and the light chain determines the specificity of the AA transporter [59,60].In ila S2 cells, a light chain called Minidiscs (MND) is required for leucine transport lobes, in the anterior part of the brain [34,44].Each MB lobe processes specific sensory inputs and thus drives particular behavioral outcomes [65].To identify the lobes in which mnd 24−1 is expressed, we used an intersectional genetic strategy combining mnd 24−1 -LexA > LexAop-FLP, and different MB-lobe-specific-Gal4 > UAS > stop > mCD8::GFP, which allowed us to identify common cells expressing mnd 24−1 -LexA and each MB-lobe-specific-Gal4 drivers.
In the adult Drosophila brain, the mushroom bodies (MBs), an integrative brain center, are a critical structure for appetitive olfactory learning and memory [25][26][27][28], and aversive olfactory memory [29,30].In addition, the integration of metabolic cues by the MBs has a critical impact on the control of learned behaviors [31][32][33].The MBs consist of 2000 to 2500 neurons per hemisphere [34], called Kenyon cells [35], which project their axons into two (α and α′) vertical lobes and three (β, β′, and ɣ) medial lobes [34,36].Kenyon cells receive olfactory inputs from the antennal lobes via projection neurons, process the information, and enable olfactory associative learning [37] and memory [38].Numerous other stimuli, such as visual, thermosensory, and gustatory inputs [39][40][41][42][43][44], are delivered to the MBs, placing this structure at the center of signal integration and behaviors.In addition, the MB α/β lobes are associated with lifespan [45], which is directly influenced by the quality and quantity of the diet and EAA intake [45].
In the adult Drosophila brain, the mushroom bodies (MBs), an integrative brain center, are a critical structure for appetitive olfactory learning and memory [25][26][27][28], and aversive olfactory memory [29,30].In addition, the integration of metabolic cues by the MBs has a critical impact on the control of learned behaviors [31][32][33].The MBs consist of 2000 to 2500 neurons per hemisphere [34], called Kenyon cells [35], which project their axons into two (α and α′) vertical lobes and three (β, β′, and ɣ) medial lobes [34,36].Kenyon cells receive olfactory inputs from the antennal lobes via projection neurons, process the information, and enable olfactory associative learning [37] and memory [38].Numerous other stimuli, such as visual, thermosensory, and gustatory inputs [39][40][41][42][43][44], are delivered to the MBs, placing this structure at the center of signal integration and behaviors.In addition, the MB α/β lobes are associated with lifespan [45], which is directly influenced by the quality and quantity of the diet and EAA intake [45].
In this study, we investigate the function of MND in the response to AAs in the adult brain.First, we show that mnd is expressed in both glial cells and neurons in the adult Drosophila brain.By generating specific Gal4 and LexA reporter transgenes, we identified two distinct regulatory regions in the mnd gene that control expression either in the mushroom body neurons (α/β and ɣ lobe neurons of the MBs) or in cortex glia.We used the MB-specific mnd driver to study the neuronal activity in response to different AAs in ex vivo functional brain imaging and revealed that MB neurons respond to a wide range of AAs.Here, we show that mnd knockdown in α/β MB lobes results in impaired responses to several AAs, including Leu, Ile, Arg, Asp, Glu, Lys, and Thr.Finally, we found that Leucine, an EAA, activates the MBs through the TOR pathway rather than a GDH signaling pathway.Our data establish that MBs are an important brain center for internal AA sensing that depends on the presence of MND, and this highlights how amino acid transporters, such as SLC family transporters, can influence neuronal activity, which may provide new clues to better understand the regulation of neuronal activity.
AA transporters, particularly members of the Solute Carrier family (SLC), damental role in AA transport in cells, including neurons [46][47][48].SLC trans divided into several families, particularly the SLC7A family which is divide subfamilies: the Cationic Amino-acid Transporters (CATs) [49], and the L-ty acid Transporters (LATs) [50].In Drosophila, a specific member of the CAT fam Slimfast, is expressed in larval fat body cells and in adult DANs, allowing the of TOR [51,52] and the AA sensor GCN2 [18], respectively.This activation le regulation of growth and food intake [18,51].While CATs, such as Slimfast, f monomers, LATs form Heterodimeric Amino-acid Transporters (HATs) [50 HATs consist of two subunits, a heavy chain (SLC3A2/CD98hc) and a light cha coded by five putative genes in Drosophila (minidiscs, JhI-21, genderblind, sob CG1607) [55][56][57][58].In mammals, SLC3A2/CD98/4F2hc targets the complex to t membrane, and the light chain determines the specificity of the AA transporter Drosophila S2 cells, a light chain called Minidiscs (MND) is required for leucin [61].
In this study, we investigate the function of MND in the response to AAs i brain.First, we show that mnd is expressed in both glial cells and neurons in Drosophila brain.By generating specific Gal4 and LexA reporter transgenes, we two distinct regulatory regions in the mnd gene that control expression either in room body neurons (α/β and ɣ lobe neurons of the MBs) or in cortex glia.W MB-specific mnd driver to study the neuronal activity in response to different vivo functional brain imaging and revealed that MB neurons respond to a wid AAs.Here, we show that mnd knockdown in α/β MB lobes results in impaired to several AAs, including Leu, Ile, Arg, Asp, Glu, Lys, and Thr.Finally, we Leucine, an EAA, activates the MBs through the TOR pathway rather than a G ing pathway.Our data establish that MBs are an important brain center for in sensing that depends on the presence of MND, and this highlights how amino porters, such as SLC family transporters, can influence neuronal activity, whic vide new clues to better understand the regulation of neuronal activity.

Drosophila Strains
All Drosophila melanogaster strains were maintained on standard cornmeal medium, at 25 °C, on a 12 h:12 h light-dark cycle, with 50-60% relative humid strains used in this study were as follows: w 1118 , mnd 24−1 -Gal4, mnd 25−1 -Gal4, a lobes and not in α ′ /β ′ lobes of the MBs (Figure 3(d2)).The presence of MND was confirmed by co-localization of MND and GFP (Figure 3(d1,d1 ′ ,d1 ′′ )).Thus, we have shown that the mnd 24−1 promoter sequence specifically drives expression in the α/β lobes and in the Cells 2024, 13, x FOR PEER REVIEW (IPCs) in the brain via a GDH pathway to ensure proper larval metabolism [ onine-supplemented diets reduce survival but have no effect on repro whereas reduced methionine intake extends lifespan and reduces reproducti status [23,24].Threonine, another EAA, is a sleep-promoting molecule that l metabolism to GABAergic control of sleep.Thus, threonine may be the neuro for sleep homeostasis [9].
AA transporters, particularly members of the Solute Carrier family (SLC damental role in AA transport in cells, including neurons [46][47][48].SLC tra divided into several families, particularly the SLC7A family which is divi subfamilies: the Cationic Amino-acid Transporters (CATs) [49], and the L acid Transporters (LATs) [50].In Drosophila, a specific member of the CAT fa Slimfast, is expressed in larval fat body cells and in adult DANs, allowing of TOR [51,52] and the AA sensor GCN2 [18], respectively.This activation regulation of growth and food intake [18,51].While CATs, such as Slimfas monomers, LATs form Heterodimeric Amino-acid Transporters (HATs) [ HATs consist of two subunits, a heavy chain (SLC3A2/CD98hc) and a light c coded by five putative genes in Drosophila (minidiscs, JhI-21, genderblind, s CG1607) [55][56][57][58].In mammals, SLC3A2/CD98/4F2hc targets the complex t membrane, and the light chain determines the specificity of the AA transpor Drosophila S2 cells, a light chain called Minidiscs (MND) is required for leuc [61].
In this study, we investigate the function of MND in the response to AA brain.First, we show that mnd is expressed in both glial cells and neuron Drosophila brain.By generating specific Gal4 and LexA reporter transgenes, two distinct regulatory regions in the mnd gene that control expression either room body neurons (α/β and ɣ lobe neurons of the MBs) or in cortex glia.MB-specific mnd driver to study the neuronal activity in response to differe vivo functional brain imaging and revealed that MB neurons respond to a w AAs.Here, we show that mnd knockdown in α/β MB lobes results in impair to several AAs, including Leu, Ile, Arg, Asp, Glu, Lys, and Thr.Finally, w Leucine, an EAA, activates the MBs through the TOR pathway rather than a ing pathway.Our data establish that MBs are an important brain center fo sensing that depends on the presence of MND, and this highlights how ami porters, such as SLC family transporters, can influence neuronal activity, wh vide new clues to better understand the regulation of neuronal activity.
Cells 2024, 13, x FOR PEER REVIEW 9 of 18  (IPCs) in the brain via a GDH pathway to ensure proper larval metabolism [20,21].Methi onine-supplemented diets reduce survival but have no effect on reproduction [22 whereas reduced methionine intake extends lifespan and reduces reproduction in low AA status [23,24].Threonine, another EAA, is a sleep-promoting molecule that links neurona metabolism to GABAergic control of sleep.Thus, threonine may be the neuronal substrat for sleep homeostasis [9].
In the adult Drosophila brain, the mushroom bodies (MBs), an integrative brain center are a critical structure for appetitive olfactory learning and memory [25][26][27][28], and aversiv olfactory memory [29,30].In addition, the integration of metabolic cues by the MBs has critical impact on the control of learned behaviors [31][32][33].The MBs consist of 2000 to 250 neurons per hemisphere [34], called Kenyon cells [35], which project their axons into tw (α and α′) vertical lobes and three (β, β′, and ɣ) medial lobes [34,36].Kenyon cells receiv olfactory inputs from the antennal lobes via projection neurons, process the information and enable olfactory associative learning [37] and memory [38].Numerous other stimul such as visual, thermosensory, and gustatory inputs [39][40][41][42][43][44], are delivered to the MBs placing this structure at the center of signal integration and behaviors.In addition, the MB α/β lobes are associated with lifespan [45], which is directly influenced by the quality and quantity of the diet and EAA intake [45].
In this study, we investigate the function of MND in the res brain.First, we show that mnd is expressed in both glial cells a Drosophila brain.By generating specific Gal4 and LexA reporter two distinct regulatory regions in the mnd gene that control expre room body neurons (α/β and ɣ lobe neurons of the MBs) or in MB-specific mnd driver to study the neuronal activity in respon vivo functional brain imaging and revealed that MB neurons re AAs.Here, we show that mnd knockdown in α/β MB lobes resu to several AAs, including Leu, Ile, Arg, Asp, Glu, Lys, and Th Leucine, an EAA, activates the MBs through the TOR pathway r ing pathway.Our data establish that MBs are an important bra sensing that depends on the presence of MND, and this highligh porters, such as SLC family transporters, can influence neuronal vide new clues to better understand the regulation of neuronal a
In this study, we investigate the function of MND in the response to AAs in brain.First, we show that mnd is expressed in both glial cells and neurons in Drosophila brain.By generating specific Gal4 and LexA reporter transgenes, we two distinct regulatory regions in the mnd gene that control expression either in room body neurons (α/β and ɣ lobe neurons of the MBs) or in cortex glia.We MB-specific mnd driver to study the neuronal activity in response to different vivo functional brain imaging and revealed that MB neurons respond to a wid AAs.Here, we show that mnd knockdown in α/β MB lobes results in impaired to several AAs, including Leu, Ile, Arg, Asp, Glu, Lys, and Thr.Finally, we f Leucine, an EAA, activates the MBs through the TOR pathway rather than a GD ing pathway.Our data establish that MBs are an important brain center for in sensing that depends on the presence of MND, and this highlights how amino a porters, such as SLC family transporters, can influence neuronal activity, which vide new clues to better understand the regulation of neuronal activity.
In this study, we investigate the function of MND in the response to A brain.First, we show that mnd is expressed in both glial cells and neuro Drosophila brain.By generating specific Gal4 and LexA reporter transgene two distinct regulatory regions in the mnd gene that control expression eith room body neurons (α/β and ɣ lobe neurons of the MBs) or in cortex gli MB-specific mnd driver to study the neuronal activity in response to diffe vivo functional brain imaging and revealed that MB neurons respond to a AAs.Here, we show that mnd knockdown in α/β MB lobes results in impa to several AAs, including Leu, Ile, Arg, Asp, Glu, Lys, and Thr.Finally, Leucine, an EAA, activates the MBs through the TOR pathway rather than ing pathway.Our data establish that MBs are an important brain center f sensing that depends on the presence of MND, and this highlights how am lobes of the MBs.For all images the scale bar is 50 µm.

MND Is Required for AA Dependent Activity of Kenyon Cells
MND is present in MB neurons, but its precise function as an AA transporter has not yet been demonstrated in vivo.MND has been described as a Leu transporter in S2 cell cultures [61] and as a Leucine sensor in larval IPCs where it enables DILP release [20].To test whether and how MND impacts neuronal activity within the MB, we expressed the calcium sensor GCaMP6s in mnd 24−1 -Gal4 expressing neurons (mnd 24−1 -Gal4 > GCaMP6s) and studied their activation using calcium imaging on ex vivo brains.In the brain, glutamate released by neurons or glial cells activates MB neurons, which express different glutamate receptors [66][67][68][69][70]. Therefore, we decided to test the activity of the mnd 24−1 -positive MB neurons in response to different concentrations of Glutamate in 3-, 6-, or 9-day-old flies.We tested Glu concentrations from 0.2 mM to 20 mM, which was previously used to activate neurons in Drosophila [71][72][73][74][75]).The highest response was observed in 3-day-old flies when 20 mM Glu was applied to the brains (Figure S2).Consequently, we tested the response to the remaining 19 natural L-AAs using the same conditions.
To elucidate the role of MND in MB activity in response to L-amino acids, we examined the activity of mnd 24−1 -Gal4 > GCaMP6s;mnd dsRNAKK brains in which mnd is knockeddown only in the mnd 24−1 positive neurons expressing the calcium sensor GCaMP6s (Figures 4b,c and S1b).These results are consistent with our previous experiments showing that MND is involved in the transport of Leu, Ile, and not Val in larval IPCs to enable the release of DILPs [20].In addition, we show that MND is also involved in the sensing of other L-AAs such as Arg, Asp, Glu, Lys, and Thr in the α/β MB neurons (Figure 4c).

The TOR Signaling Pathway Mediates the Stimulation of the MBs by Leucine
Leucine, an EAA supplied through dietary food intake, is capable of regulating cell function through either the Target Of Rapamycin (TOR) pathway or the Glutamate Dehydrogenase (GDH) pathway [20,[76][77][78][79].In a previous study, we demonstrated that Leu triggers the release of DILPs via the GDH pathway in an MND-dependent manner [20].Since Leu and Ile, two amino acids, activate the MBs that express mnd (Figure 4c), we examined the downstream GDH and TOR signaling pathways (Figure 5a).In adult brains, inactivation of GDH did not reduce the activity of the α/β MB neurons in response to L-Leu (Figure 5b).We next investigated whether MB activation by L-Leu could be mediated by the TOR pathway.Overexpression of a dominant negative form of TOR (TOR TED ) in mnd 24−1 -positive neurons impaired the L-Leu-dependent stimulation of the MBs (Figure 5b).Similarly, the mnd 24−1 -positive MB neurons are not activated by L-Leu when TOR expression is silenced by RNAi (Figure 5b).These results show that the stimulation of the α/β MB neurons by Leucine depends on MND and the TOR pathway, but not on the GDH pathway.To elucidate the role of MND in MB activity in response to L-amino acids, we examined the activity of mnd 24−1 -Gal4 > GCaMP6s;mnd dsRNAKK brains in which mnd is knockeddown only in the mnd 24−1 positive neurons expressing the calcium sensor GCaMP6s (Fig- ures 4b,c and S1b).These results are consistent with our previous experiments showing that MND is involved in the transport of Leu, Ile, and not Val in larval IPCs to enable the release of DILPs [20].In addition, we show that MND is also involved in the sensing of other L-AAs such as Arg, Asp, Glu, Lys, and Thr in the α/β MB neurons (Figure 4c).

The TOR Signaling Pathway Mediates the Stimulation of the MBs by Leucine
Leucine, an EAA supplied through dietary food intake, is capable of regulating cell function through either the Target Of Rapamycin (TOR) pathway or the Glutamate Dehydrogenase (GDH) pathway [20,[76][77][78][79].In a previous study, we demonstrated that Leu triggers the release of DILPs via the GDH pathway in an MND-dependent manner [20].Since Leu and Ile, two amino acids, activate the MBs that express mnd (Figure 4c), we examined the downstream GDH and TOR signaling pathways (Figure 5a).In adult brains, inactivation of GDH did not reduce the activity of the α/β MB neurons in response to L-Leu (Figure 5b).We next investigated whether MB activation by L-Leu could be mediated by the TOR pathway.Overexpression of a dominant negative form of TOR (TOR TED ) in mnd 24−1 -positive neurons impaired the L-Leu-dependent stimulation of the MBs (Figure 5b).Similarly, the mnd 24−1 -positive MB neurons are not activated by L-Leu when TOR expression is silenced by RNAi (Figure 5b).These results show that the stimulation of the α/β MB neurons by Leucine depends on MND and the TOR pathway, but not on the GDH pathway. of ex vivo brains expressing a calcium sensor in the α/β lobes of the MBs in control brains (mnd 24−1 -Gal4 > GCaMP6s), in GDH-downregulated brains (mnd 24−1 -Gal4 > GCaMP6s; UAS-GDH dsRNAkk ), or in TOR downregulated brains (mnd 24−1 -Gal4 > GCaMP6s; UAS-TOR TED or mnd 24−1 -Gal4 > GCaMP6s; UAS-TOR dsRNA ) of 3-day-old flies exposed to L-Leu at a concentration of 20 mM.Each histogram represents the averaged fluorescence intensity of peaks ± SEM in α/β lobes of the MBs.All individual data are shown by dots (n = 9 to 13).All data were compared with the control (mnd 24−1 -Gal4 > GCaMP6s) by a Mann-Whitney test.ns: not significant, ***: p < 0.001; ****: p < 0.0001.

mnd Is Expressed in Different Cell Types in the Adult Brain
In this study, we show that MND, a LAT1-like AA transporter, is present in the adult Drosophila brain.Our data reveal the expression of two mnd mRNAs, mnd-RA and mnd-RC, within the Drosophila head, while the third predicted mnd-RD transcript was undetectable, indicating either its absence or very low expression levels.Furthermore, we have shown that MND is expressed by both neurons and glia, suggesting that mnd may be regulated by distinct regulatory sequences in the mnd promoter.By examining the promoter regulatory region of mnd, we show that two specific regulatory sequences drive expression in two different regions of the adult brain.mnd 25−1 -Gal4 drives MND expression specifically in cortex glial cells.These findings are consistent with previous studies showing that mnd is expressed in glial cells [75,80].We observe that mnd 24−1 -Gal4 is expressed in MB neurons.Our intersectional genetic strategies reveal that the mnd 24−1 regulatory sequence drives expression in the α/β and , x FOR PEER REVIEW 2 of 18 (IPCs) in the brain via a GDH pathway to ensure proper larval metabolism [20,21].Methionine-supplemented diets reduce survival but have no effect on reproduction [22], whereas reduced methionine intake extends lifespan and reduces reproduction in low AA status [23,24].Threonine, another EAA, is a sleep-promoting molecule that links neuronal metabolism to GABAergic control of sleep.Thus, threonine may be the neuronal substrate for sleep homeostasis [9].
In the adult Drosophila brain, the mushroom bodies (MBs), an integrative brain center, are a critical structure for appetitive olfactory learning and memory [25][26][27][28], and aversive olfactory memory [29,30].In addition, the integration of metabolic cues by the MBs has a critical impact on the control of learned behaviors [31][32][33].The MBs consist of 2000 to 2500 neurons per hemisphere [34], called Kenyon cells [35], which project their axons into two (α and α′) vertical lobes and three (β, β′, and ɣ) medial lobes [34,36].Kenyon cells receive olfactory inputs from the antennal lobes via projection neurons, process the information, and enable olfactory associative learning [37] and memory [38].Numerous other stimuli, such as visual, thermosensory, and gustatory inputs [39][40][41][42][43][44], are delivered to the MBs, placing this structure at the center of signal integration and behaviors.In addition, the MB α/β lobes are associated with lifespan [45], which is directly influenced by the quality and quantity of the diet and EAA intake [45].
In this study, we investigate the function of MND in the response to AAs in the adult brain.First, we show that mnd is expressed in both glial cells and neurons in the adult Drosophila brain.By generating specific Gal4 and LexA reporter transgenes, we identified two distinct regulatory regions in the mnd gene that control expression either in the mushroom body neurons (α/β and ɣ lobe neurons of the MBs) or in cortex glia.We used the MB-specific mnd driver to study the neuronal activity in response to different AAs in ex vivo functional brain imaging and revealed that MB neurons respond to a wide range of AAs.Here, we show that mnd knockdown in α/β MB lobes results in impaired responses to several AAs, including Leu, Ile, Arg, Asp, Glu, Lys, and Thr.Finally, we found that Leucine, an EAA, activates the MBs through the TOR pathway rather than a GDH signaling pathway.Our data establish that MBs are an important brain center for internal AA sensing that depends on the presence of MND, and this highlights how amino acid transporters, such as SLC family transporters, can influence neuronal activity, which may provide new clues to better understand the regulation of neuronal activity.

Materials and Methods
lobes but not in the α ′ /β ′ lobes of the MBs.Immunostaining of MND showed that MND is located in the neurons of the α/β and Cells 2024, 13, x FOR PEER REVIEW (IPCs) in the brain via a GDH pathway to ensure proper larval metabolis onine-supplemented diets reduce survival but have no effect on re whereas reduced methionine intake extends lifespan and reduces reprod status [23,24].Threonine, another EAA, is a sleep-promoting molecule th metabolism to GABAergic control of sleep.Thus, threonine may be the n for sleep homeostasis [9].
In this study, we investigate the function of MND in the response to brain.First, we show that mnd is expressed in both glial cells and neu Drosophila brain.By generating specific Gal4 and LexA reporter transgen two distinct regulatory regions in the mnd gene that control expression ei room body neurons (α/β and ɣ lobe neurons of the MBs) or in cortex g MB-specific mnd driver to study the neuronal activity in response to di vivo functional brain imaging and revealed that MB neurons respond to AAs.Here, we show that mnd knockdown in α/β MB lobes results in im to several AAs, including Leu, Ile, Arg, Asp, Glu, Lys, and Thr.Finall Leucine, an EAA, activates the MBs through the TOR pathway rather th ing pathway.Our data establish that MBs are an important brain cente sensing that depends on the presence of MND, and this highlights how porters, such as SLC family transporters, can influence neuronal activity vide new clues to better understand the regulation of neuronal activity.lobes but also in the α ′ /β ′ lobes of the MBs, suggesting the mnd 24−1 regulatory sequence only controls the expression of mnd in a subset of neurons of the MBs and implying that another neuronal regulatory sequence of mnd exists.

AAs Stimulate Neuronal Activity in the MBs
The EAAs Leu, Ile, and Thr must be supplied by the diet and they can act as nutrient signals to regulate the state of nutritional homeostasis [9,20,81].Interestingly, the MBs are an integrated center for hunger control of food-seeking behaviour receiving input signals of hunger and satiety [33].DANs regulate MB activity and control innate olfactory behaviour.These DANs are under the control of satiety signals such as insulin-like peptides and AstA or hunger signals such as NPF, sNPF, and serotonin [33].Here, we show that the α/β lobes of the MBs are activated by EAAs such as Leu, Ile, and Thr.Thus, EAAs, nutrients arriving from the gut, can directly or indirectly affect neuronal functions driven by the MBs.We also show that Glu, an excitatory neurotransmitter, significantly enhances the activity of the MBs.The increase in calcium activity in the MBs induced by Glu may be due to a direct or indirect action of Glu.

Disruption of mnd Impairs Response of the MBs to AAs
Initially, mnd was described to be involved in the non-autonomous development of imaginal discs in the larvae [55].We did not observe any changes in the shape and length of the α/β lobes when mnd was downregulated in the MBs, suggesting that MND is not involved in the development of the MBs [82].In our experiments, the downregulation of mnd in the MBs does not reduce neuronal activity in response to amino acids such as Ala, Asp, Gly, His, Phe, Pro, Tyr, and Val (Figure 4c).Disruption of MND function in the α/β lobes of the MBs reduces neuronal activity in the response to certain AAs, such as Leu and Ile.In mammals, Leucine regulates the activity of hypothalamic POMC neurons to control food intake [83] through mTOR signaling [84].In Drosophila, MBs integrate hunger and food signals to monitor food-seeking behaviour [33].We show that MBs are sensitive to Leu suggesting that MBs integrate protein satiety signals to adjust their activity.Previously, we have shown that MND is required for the sensing of Leu in the larval brain IPCs, resulting in an increase of neuronal activity and, DILPs release through a GDH pathway [20,21].

Figure 1 .
Figure 1.mnd is expressed in the adult Drosophila brain.(a) Schematic representation of the structure of the mnd gene, located on the left arm of the third chromosome (14,985,100-14,991,667 bp).Exons from 1 to 5 are represented by orange boxes and introns by black lines.Three mRNA isoforms were predicted by the ORF analysis from expasy.org:mnd-RD (in blue), mnd-RC (in purple), and mnd-RA (in gray).The 5′ and 3′ untranslated regions are indicated by black boxes.The arrows under the transcripts indicate the primers used for the RT-PCR analysis: green for the common region of all three alternative mnd mRNAs; gray for mnd-RA; purple for mnd-RC; and blue for mnd-RD.Scale bar, 1 kb.(b) mnd expression analyzed by RT-PCR using RNAs extracted from w 1118 adult heads and bodies.PCR products were analyzed by electrophoresis on agarose gel.mnd-RC (183 pb) and mnd-RA (111 pb) are both present in the adult heads and bodies, whereas mnd-RD (114 pb) was not detected in heads or bodies.Primers used for RT-PCR are indicated in (a) with colored arrows: green for the common portion of the three alternative mnd mRNAs; gray for mnd-RA; purple for mnd-RC; and blue for mnd-RD.(c) Representative images of double immunostaining with anti-MND (magenta) and anti-GFP (green) on elav-Gal4 > UAS-mCD8::GFP brain (neuronal marker) or on repo-Gal4 > UAS-mCD8::GFP brain (glial cell marker).Red boxes illustrate the area of the MB calyx (upper image) and the cortex glia (lower image) in the brain where the images were recorded.A total of 12 brains for each condition were examined.MND is present in both neurons and glial cells in the adult brain.White indicates the overlap of the two markers on merged images.Scale bar, 50 µm.

Figure 1 .
Figure 1.mnd is expressed in the adult Drosophila brain.(a) Schematic representation of the structure of the mnd gene, located on the left arm of the third chromosome (14,985,100-14,991,667 bp).Exons from 1 to 5 are represented by orange boxes and introns by black lines.Three mRNA isoforms were predicted by the ORF analysis from expasy.org:mnd-RD (in blue), mnd-RC (in purple), and mnd-RA (in gray).The 5 ′ and 3 ′ untranslated regions are indicated by black boxes.The arrows under the transcripts indicate the primers used for the RT-PCR analysis: green for the common region of all three alternative mnd mRNAs; gray for mnd-RA; purple for mnd-RC; and blue for mnd-RD.Scale bar, 1 kb.(b) mnd expression analyzed by RT-PCR using RNAs extracted from w 1118 adult heads and bodies.PCR products were analyzed by electrophoresis on agarose gel.mnd-RC (183 pb) and mnd-RA (111 pb) are both present in the adult heads and bodies, whereas mnd-RD (114 pb) was not detected in heads or bodies.Primers used for RT-PCR are indicated in (a) with colored arrows: green for the common portion of the three alternative mnd mRNAs; gray for mnd-RA; purple for mnd-RC; and blue for mnd-RD.(c) Representative images of double immunostaining with anti-MND (magenta) and anti-GFP (green) on elav-Gal4 > UAS-mCD8::GFP brain (neuronal marker) or on repo-Gal4 > UAS-mCD8::GFP brain (glial cell marker).Red boxes illustrate the area of the MB calyx (upper image) and the cortex glia (lower image) in the brain where the images were recorded.A total of 12 brains for each condition were examined.MND is present in both neurons and glial cells in the adult brain.White indicates the overlap of the two markers on merged images.Scale bar, 50 µm.

Figure 2 .
Figure 2. mnd regulatory sequences lead to different expression patterns of MND.mnd regulatory sequences lead to different MND expression patterns, and mnd 24−1 tools mimic a part of mnd

Figure 3 .
Figure 3. mnd is expressed in Kenyon cells forming α/β and

Figure 4 .
Figure 4. MND is required for AA sensing in the MBs.Real-time calcium imaging of ex vivo brains expressing a calcium sensor in the α/β lobes of the MBs in control brains (mnd 24−1 -Gal4 > GCaMP6s) or in mnd downregulated brains (mnd 24−1 -Gal4 > GCaMP6s;UAS-mnd dsRNAkk ) of 3-day-old flies exposed to each L-AA at a concentration of 20 mM.The different intensities of basal GCaMP6s levels in the vicinity of the two lobes are sometimes difficult to convert to similar rainbow colors, and thus in some brains only one lobe appears in false color.(a) Representative images, in false colors, showing the fluorescence level before (basal activity) and after the addition of either control Ringer's solution or Glu (20 mM) in control brains (mnd 24−1 -Gal4 > GCaMP6s).Scale bar, 50 µm.Line plots of fluorescence changes (∆F/F0) in α/β lobe neurons stimulated with Ringer's solution or 20 mM of Glu for one representative brain.Stimulus application is indicated by a red arrow.(b) Representative images, in false colors, showing the fluorescence level before (basal activity) and after the addition of either control Ringer's solution or Glu (20 mM) in mnd downregulated brains (mnd 24−1 -Gal4 > GCaMP6s;UAS-mnd dsRNAkk ).Scale bar, 50 µm.Line plots of fluorescence changes (∆F/F0) in α/β lobe

Figure 4 .
Figure 4. MND is required for AA sensing in the MBs.Real-time calcium imaging of ex vivo brains expressing a calcium sensor in the α/β lobes of the MBs in control brains (mnd 24−1 -Gal4 > GCaMP6s) or in mnd downregulated brains (mnd 24−1 -Gal4 > GCaMP6s;UAS-mnd dsRNAkk ) of 3-day-old flies exposed to each L-AA at a concentration of 20 mM.The different intensities of basal GCaMP6s levels in the vicinity of the two lobes are sometimes difficult to convert to similar rainbow colors, and thus in some brains only one lobe appears in false color.(a) Representative images, in false colors, showing the fluorescence level before (basal activity) and after the addition of either control Ringer's solution or Glu (20 mM) in control brains (mnd 24−1 -Gal4 > GCaMP6s).Scale bar, 50 µm.Line plots of fluorescence changes (∆F/F 0 ) in α/β lobe neurons stimulated with Ringer's solution or 20 mM of Glu

Figure 5 .Figure 5 .
Figure 5. Leucine-mediated activity is TOR-dependent in the MBs.(a) Schematic representation of a Kenyon cell and the putative pathways downstream of leucine activity.(b) Real-time calcium imaging of ex vivo brains expressing a calcium sensor in the α/β lobes of the MBs in control brains (mnd 24−1 -Gal4 > GCaMP6s), in GDH-downregulated brains (mnd 24−1 -Gal4 > GCaMP6s; UAS-Figure 5. Leucine-mediated activity is TOR-dependent in the MBs.(a) Schematic representation of a Kenyon cell and the putative pathways downstream of leucine activity.(b) Real-time calcium imaging Cells 2024, 13, x FOR PEER REVIEW 12 of 18 neurons stimulated with Ringer's solution or 20 mM of Glu for one representative brain.Stimulus application is indicated by a red arrow.(c) Averaged fluorescence intensity of positive or negative peaks ± SEM for control brains (mnd 24−1 -Gal4 > GCaMP6s, blue histograms) and for mnd downregulated brains (mnd 24−1 -Gal4 > GCaMP6s;UAS-mnd dsRNAkk , orange histograms) in response to either Ringer's solution (Ctl) or a specific L-AA at 20 mM.All individual data are shown by dots (n = 10 to 23).For each L-AA, data obtained from mnd downregulated brains (mnd 24−1 -Gal4 > GCaMP6s; UASmnd dsRNAkk ) were compared to the corresponding control (mnd 24−1 -Gal4 > GCaMP6s) using a Mann-Whitney test.The absence of * for a given AA indicates that the data are not statistically different between the two conditions.*: p < 0.05 ; **: p < 0.01 ; ***: p < 0.001 ; ****: p < 0.0001.